Patatin-like phospholipase CapV in Escherichia coli - morphological and physiological effects of one amino acid substitution

In rod-shaped bacteria, morphological plasticity occurs in response to stress, which blocks cell division to promote filamentation. We demonstrate here that overexpression of the patatin-like phospholipase variant CapVQ329R, but not CapV, causes pronounced sulA-independent pyridoxine-inhibited cell filamentation in the Escherichia coli K-12-derivative MG1655 associated with restriction of flagella production and swimming motility. Conserved amino acids in canonical patatin-like phospholipase A motifs, but not the nucleophilic serine, are required to mediate CapVQ329R phenotypes. Furthermore, CapVQ329R production substantially alters the lipidome and colony morphotype including rdar biofilm formation with modulation of the production of the biofilm activator CsgD, and affects additional bacterial traits such as the efficiency of phage infection and antimicrobial susceptibility. Moreover, genetically diverse commensal and pathogenic E. coli strains and Salmonella typhimurium responded with cell filamentation and modulation in colony morphotype formation to CapVQ329R expression. In conclusion, this work identifies the CapV variant CapVQ329R as a pleiotropic regulator, emphasizes a scaffold function for patatin-like phospholipases, and highlights the impact of the substitution of a single conserved amino acid for protein functionality and alteration of host physiology.


Supplementary results
The dinucleotide cyclase DncV synthesizes cAMP-GMP to inhibit rdar biofilm formation and motility in the animal commensal strain E. coli ECOR31 (1). DncV is flanked by V. cholerae 7 th pandemic island-1 (VSP-1) homologs; capV upstream and vc0180 and vc0181 downstream (1-3) ( Supplementary Fig. 1a). These genes constitute a 4.6 kbp four-gene cluster which contributes to differential functionality of dncV in E. coli ECOR31 (1). To understand physiological functions of the putative four-gene operon (designed 78901), we had previously cloned the four genes in pBAD28 under the regulation of the pBAD promoter (1). In this work, we tested their combined effect on swimming motility in semi-solid agar for the heterologous host E. coli K-12 derivative MG1655. Overexpression of the cloned four-gene operon (designated 78901v, see below) from the pBAD promoter in the pBAD28 plasmid significantly inhibited swimming motility at 37 °C and 28 °C, as did five other independent clones (Supplementary Fig. 1b and c and data not shown). Since the plate assay monitors both chemotaxis and motility, we assessed production of cell associated flagellin FliC upon overexpression of 78901v. While E. coli MG1655 pronouncedly produced flagellin, which stems from flagella, polymerized monomeric flagellin, overexpression of 78901v inhibited flagellin production ( Supplementary Fig. 1d). The first ORF of capV-dncV-vc0180-vc0181 is capV ( Supplementary Fig. 1a). CapV possesses a N-terminal patatin-like phospholipase A2 (PNPLA) domain, the enzymatic activity of which is stimulated by cAMP-GMP in V. cholerae (4). VC0180 encodes an eukaryotic-like ubiquitin ligase with ThiF (E1)/ E2 domains and VC0181, an isopeptidase with a JAB domain, respectively (3, 4) ( Supplementary Fig. 2a). To clarify which gene in the putative four-gene operon contributes to inhibition of E. coli MG1655 swimming motility, we constructed amino acid substitutions in the catalytic motif of each of the gene products. Expression of the four genes from p78901v (Table S1) with an amino acid substitution in the D-G-[A/G] motif of CapV (leading to 78901vD197A) did not suppress apparent swimming motility nor cell-associated and secreted flagellin production, while substitutions in residues involved in the catalysis of other proteins maintained the suppressive effect ( Supplementary Fig. 1d and 2b-e). Surprisingly, both overexpression of wild type capV and dncV*-vc0180-vc0181* (p79801v) had no effect on apparent swimming motility of E. coli MG1655 ( Supplementary Fig. 3a). Subsequently, sequence analysis showed the presence of six mutations in the cloned insert of p78901v, whereby the nucleotide alternation in the capV open reading frame led to the substitution of glutamine 329 by arginine (Q329R) (Supplementary Fig. 3a and c). Three additional nucleotide alterations provide non-synonymous mutations (besides Q329R, leading to K102R and L275P in DncV and, putatively, R6K in VC0181). To investigate the molecular basis of apparent swimming repression of p78901v, we constructed a plasmid that expressed the CapVQ329R variant, which suppressed apparent swimming motility. Moreover, only reversion of the Q329R mutation in construct p78901v expressing the four genes resulting to p78901vR329Q relieved swimming repression, while nucleotide substitutions reverting the non-synonymous mutations in dncV and vc0181 failed to relieve swimming repression ( Supplementary Fig. 3b, d, e).

Supplementary Figures
Supplementary Figure 1. Overexpression of the variant four gene operon capV-dncV-vc0180-vc0181 cloned in pBAD28 (p78901v) (a) downregulates swimming motility (b and C) and FliC production (d) of E. coli MG1655. Three µl of OD600 = 5 cells were inoculated into soft agar plates containing 1% tryptone, 0.5% NaCl and 0.25% agar and the swimming diameter was measured after 8 h at 28 °C and 6 h at 37 °C. Bars represent mean values with error bars to represent standard deviation (SD) from three independent replicates. Differences between mean values were assessed by two-tailed Student's t-test (**p < 0.01, and ***p < 0.001 compared to MG1655 vector control). VC = pBAD28, p78901v = capVQ329R-dncV*-vc0180-vc0181* cloned in pBAD28, p78901vD197A = capVQ329R/D197A-dncV*-vc0180-vc0181* cloned in pBAD28. Figure 2. CapVQ329R of p78901v, but not other variant gene products encoded by p78901v downregulate apparent swimming motility of MG1655 a Schematic illustration of E. coli ECOR31 four gene operon with domain profiles of gene products. The putative catalytic residues are indicated by filled red triangles. Domain prediction was performed with the InterPro server, and the result was processed with ExPASy_Prosite_-MyDomains. b-e Effects of aa substitutions in the catalytic motifs of p78901v encoded gene products on apparent swimming motility of E. coli MG1655 Three µl of OD600 = 5 cells were inoculated into soft agar plates containing 1% tryptone, 0.5% NaCl and 0.25% agar and the swimming diameter was measured after 6 h at 37 °C. VC = pBAD28, p78901v = capVQ329R-dncV*-vc0180-vc0181* cloned in pBAD28. pCapV = CapV cloned in pBAD28;

Supplementary Figure 3. CapVQ329 production is critical for induction of apparent inhibition of swimming motility by p78901v or expressed as a stand-alone gene product in E. coli
MG1655. a and b, Effects of the aa substitution in CapV upon dissection of the p78901v construct on apparent swimming motility of MG1655. p78901v represents capVQ329R-dncV*-vc0180-vc0181*; p79801v represents dncV*-vc0180-vc0181* cloned in pBAD28. pCapV = CapV cloned in pBAD28. Q329R = CapVQ329R cloned in pBAD28. c Schematic illustration of the aa changes in the p78901v gene products compared to 78901 chromosomal sequence. d and e Effects of the aa substitution in DncV* and VC0181* on functionality of the p78901v construct for apparent swimming motility of E. coli MG1655. R329Q = capV-dncV*-vc0180-vc0181* cloned in pBAD28.
Three µl of OD600 = 5 cell suspension was inoculated into soft agar plates containing 1% tryptone, 0.5% NaCl and 0.25% agar and the swimming diameter was measured after 6 h at 37 °C. Bars represent mean values with error bars to represent SD from three independent replicates. Differences between mean values were assessed by two-tailed Student's t-test (ns, not significant; *p < 0.05, **p < 0.01, and ***p < 0.001 compared to E. coli MG1655 vector control). VC = pBAD28. Completely conserved residues are shown in white on a red background. Conserved residues are boxed. The putative catalytic residues of CapV are indicated with filled red triangles. CapVQ329 is marked with a black asterisk above the sequence. The residues in CapVQ329R mutated to alanine are marked with red asterisks above the sequence. The consensus sequence below the alignment indicates in uppercase residues with 100% conservation and in lowercase residues higher than >70% conservation. Alignment was performed using CLUSTALW using standard parameters (5), and the result processed with ESPript 3.0 (6). b Maximum likelihood phylogenetic reconstruction of CapV homologs. CapV from E. coli ECOR31 was used as the query in the NCBI Blast search and representative proteins (one representative per genus of equal homology) with >60% aa identity over the entire length of the protein sequence were retrieved. PG, phosphatidylglycerol; PE, phosphatidylethanolamine; and FA, fatty acids derivatives by untargeted CSH-QTOF MS analysis. Bars represent mean values from five independent replicates with error bars to represent SD. Differences between mean values were assessed by two-tailed Student's t-test (ns, not significant; *p < 0.05, **p < 0.01, and ***p < 0.001 compared to MG1655 VC). Figure 6. CapVQ329R-induced cell filamentation is independent of the SOS gene sulA and does hardly affect FtsZ and FtsA production. a Cells were grown for 4 h at 37 °C and placed on an agarose pad to be observed by fluorescence microscopy. Septa are indicated by white arrows. Bar, 3 µm. b Chromosomal segregation is impaired in filamenting cells. Cells were cultured in TB medium at 37 °C for 4 h, stained with DAPI and assessed under fluorescence microscopy immediately. Large fragments of unsegregated nucleoids are indicated by white arrows. Suspected septa are indicated by orange arrows. Large spaces between nucleoids are indicated by red arrows. Bar, 5 µm. c Cells expressing FtsZ-GFP protein were grown in TB medium at 37 °C, and samples were harvested after 2 h, 3 h, and 4 h of growth for Western blot analysis. d CapVQ329R-induced cell filamentation is sulA-independent. Light microscopy pictures of cell filamentation in an E. coli MG1655 ΔsulA mutant after 4 h incubation at 37 °C imaged by light microscopy. Bar, 5 µm. e Time-lapse analysis of mCherry-MinC expressing cells (PB318) upon CapVQ329R overexpression (see also Fig. 5c). A representative elongating cell is displayed. Graphs on the right of the fluorescence images display the line profiles of fluorescent signals emanating from the cell. Arbitrary fluorescent units are obtained, analyzed by the Fiji ImageJ 1.8.0 software and plotted on the y-axis; cell length in µm is plotted on the x-axis. Bar, 3 µm. VC = pBAD28; CapV = wild type CapV cloned in pBAD28; CapVQ329R = mutant CapVQ329R cloned in pBAD28.

Supplementary Figure 7. Overproduction of CapV and CapVQ329R in E. coli MG1655 restricts cell viability as assessed by the LIVE/DEAD™ BacLight™ Viability Kit.
Cells were incubated for 3 h and 6 h at 37 °C and stained with SYTO 9 and propidium iodide (PI). The cell viability was analyzed under a fluorescence microscope. Green, SYTO9 uptake/live cells. Red, PI uptake/dead cells. Blue arrows, uptake of SYTO9 and PI. Bar, 5 µm. VC = pBAD28; CapV = wild type capV cloned in pBAD28; CapVQ329R = mutant CapVQ329R cloned in pBAD28.

Supplementary Figure 8. Effects of various B-vitamins on CapVQ329R-induced cell filamentation of E. coli MG1655.
Addition of Vitamin B1 (thiamine, 0.5%), B2 (riboflavin, 0.5%), B3 (nicotinamide, 0.5%), B5 (pantothenate, 15 mg/ml), B7 (biotin, 100 ug/ml), B9 (folic acid, 80 ug/ml), and B12 (cobalamin, 200 ug/ml) in TB does not affect cell filamentation of E. coli MG1655 induced by CapVQ329R after 4 h at 37 °C. Bar, 10 µm. VC = pBAD28; CapV=wild type capV cloned in pBAD28; CapVQ329R = mutant CapVQ329R cloned in pBAD28. E. coli strains and S. typhimurium UMR1. a, b 3 12 and CFT073 and the gastrointestinal pathogen S. typhimurium UMR1 were tested. e Rdar biofilm and CsgD expression of ECOR31 upon overexpression of CapV and CapVQ329R. Cells were grown on salt-free LB agar plates at 37 °C, observed after 24 h and collected for CsgD expression detection by Western blot analysis after 16 h (addition of Congo red to the medium delays the development of the phenotype). As the loading control, the blot was stained with ponceau S after antigen detection. f CsgD expression of E. coli MG1655 upon overexpression of CapV and CapVQ329R. Cells were grown on salt-free LB agar plates at 37 °C for 40 h and collected for CsgD expression detection by Western blot analysis. PC = E. coli TOB1 as a positive control for CsgD expression. As the loading control, the blot was stained with ponceau S after antigen detection. (e and f) VC = pBAD28; pCapV = wild type capV cloned in pBAD28; Q329R = mutant CapVQ329R cloned in pBAD28.